The invention relates to gene therapy episomal expression cassettes to express a transgene in epithelial cells.
Demonstration of the feasibility of gene transfer to humans by a number of clinical trials stimulated considerable interest in gene therapy in the scientific community even though no therapeutic benefit has yet been offered to patients (7). Epithelial tissue, particularly lung epithelial tissue, has considerable potential as a target for gene therapy. The lung is a highly suitable organ for in vivo gene therapy treatment of patients with potentially lethal lung disorders, such as cystic fibrosis, cancers of epithelial origin and emphysema because of its large accessible epithelial and endothelial surface area (15). Both virus-based and non-virus-based methods can be used to deliver genes to lungs (6, 15). The use of liposomes as gene transfer agents seems to have some significant advantages for in vivo lung gene therapy (6, 15). First, liposomes offer a wide margin of safety with low toxicity and have already been used to deliver drugs to humans. They can be administered into the lungs as an aerosol, by direct lavage or following intravenous injection. A clinical trial in nasal epithelia showed no adverse effects; nasal biopsies showed no immuno-histological changes (4). Secondly, liposome-complexed DNA can be used to transfect both resting and dividing cells. In addition, large DNA constructs can be accommodated with liposomes for transfection. Finally and most importantly, liposome-mediated gene expression is episomal, thereby avoiding or reducing the risk of random chromosomal insertions. However, one of the major impediments to liposome-mediated in vivo gene therapy is that the currently available expression vectors only offer a very low level of transient transgene expression (15). Therefore, enhancement of the therapeutic gene expression would not only increase the efficacy, but also effectively decrease the already low levels of toxicity by reducing the dose of therapeutic reagent.
The inefficient expression of transgenes in lung is, at least in part, due to the lack of proper lung-specific gene expression cassettes (15). An ideal expression cassette for human lung gene therapy should be safe and confer an appropriate level of tissue-specific expression for a reasonable duration. The rational design of expression cassettes for lung gene therapy relies on our knowledge of regulation of gene expression. Regulation of eukaryotic gene expression is a very complicated process.
A particular gene may be expressed in only one type of cell or tissue while others are expressed in most cell types or tissues. For example, cytokeratin genes are expressed predominantly in epithelial cells (26). In contrast, genes encoding proteins involved in translation (protein synthesis) are expressed in every cell type. The activity of a eukaryotic gene can be regulated at any stage during the course of its expression, such as transcription, RNA splicing, RNA stability, translation, or post-translational modification. Current knowledge indicates that transcription and RNA splicing are the major steps for regulation of many eukaryotic genes.
1.2a Transcriptional regulation
Transcription of eukaryotic genes is catalyzed by an RNA polymerase which is recruited to the promoter by multiple protein factors involved in transcription initiation. Regulation of transcription can be attributed to tissue-specific DNA elements (enhancers or silencers) that stimulate or repress transcription through interaction with tissue-specific transcription factors (25). However, these elements may not function if they reside in an inappropriate location on a chromosome, suggesting that chromosomal position and structure also affect gene expression. This has led to discovering a type of regulatory elements called locus control region (LCR) (13). These LCRs, when integrated into chromosomes, confer copy number-dependent and location-independent gene expression. The first LCR was discovered in 5xe2x80x2 region of the human xcex2-globin gene cluster (9, 10, 13). LCRs are now known to be associated with other genes (28, 36) including human cytokeratin 18 and rat LAP (C/EBPb) which direct gene expression in lung cells of transgenic mice (28, 36). Although currently there is no evidence to show that LCRs enhance episomal gene expression, this possibility can not be ruled out since information about the interactions of LCRs with other regulatory elements is still limited. If LCRs increase gene expression, they would be useful in the design of episomal expression cassettes. As lung epithelial cells are not actively dividing, the delivered plasmid DNA may be wrapped by histones or other nuclear factors and kept in a transcriptionally inactive conformation. Although it is generally believed that plasmids when transferred into nucleus do not form chromatin structures, recent experiments by Jeong and Stein demonstrate that some of the transfected DNAs are in chromatin form (17). The presence of a functional LCR in expression cassettes may allow a plasmid to stay in an open conformation.
1.2b Regulation through RNA processing
Regulation of RNA splicing is also very important for tissue-specific and developmentally regulated gene expression (35). This type of regulated RNA splicing or alternative RNA splicing can lead to the production of different proteins from a single gene by inclusion of different exons in different mRNAs. Some introns contain strong enhancers and their exclusion from expression constructs would lead to diminished gene expression. For example, the first intron of the human cytokeratin 18 contains a strong enhancer which is required for expression of the cytokeratin 18 gene (29). Other introns that do not contain enhancers may also affect gene expression. For example, the presence of rpL32 intron 3 leads to a 30-fold increase in mRNA level relative to the intronless rpL32 minigene (21). However, different introns clearly have different effects. For instance, inclusion of intact thymidylate synthase gene intron 4 alone at its normal position in the thymidylate synthase (TS) coding region leads to a decrease in the level of expression relative to that observed with a the intronless TS minigene (21). The details of this splicing regulation of expression are unknown.
Efficient tissue-specific gene expression can be achieved, in theory, by using tissue-specific promoters, promoter elements, RNA processing signals, and tissue-specific RNA-stabilizing elements. Cell-specific gene expression primarily results from either tissue-specific promoters, and/or tissue-specific regulatory elements, such as enhancers, silencers, and locus control regions (LCRs). However, it is very difficult to design a cassette for lung gene therapy because there is not enough information known about regulation of lung gene expression. Currently, no suitable expression vector for lung gene therapy has been reported. There is a pressing need for an effective expression vector because a number of human CF gene therapy trials have been conducted (7). The SV40 promoter was used to direct CFTR expression in the clinical trial by Caplen et al. (4); we observed that SV40 promoter is not very active even in cultured lung epithelial cells (see FIG. 5) and its expression in rat lung primary cells (the primary cells are first generation cells isolated from the rat lung, i.e. they are not immortalized cell lines cultured for many generations) is undetectable (Plumb and Hu, unpublished results). That might explain the large amounts of plasmid DNA (10 mg to 300 mg/per nostril) used in the study (4). Recently, several cis-acting elements and trans-acting factors regulating lung epithelial gene expression have been identified. The promoters of the SP-A (surfactant protein A), SP-B (surfactant protein B), SP-C (surfactant protein C), SP-D (surfactant protein D) and CC10 (Clara cell 10 kD protein) genes have been extensively analyzed (22, 31, 40, 41). Because these genes are predominately expressed in type II or Clara cells (22), their promoters, unless modified, would not likely be suitable for expressing genes in epithelial cells of conducting airways, which represent the primary target for CF lung gene therapy.
Because of the low efficiency in liposome-mediated gene expression, strong viral promoters are often used in gene therapy studies. However, this may not be the ideal approach for liposome-mediated lung gene therapy. For example, the CMV major immediate early gene promoter has been shown to be very strong for transient expression of transgenes in cultured cells, but two studies have shown it to be a poor promoter for lung gene expression in transgenic mice (1, 33). There is no evidence to show the CMV promoter can confer sustained episomal gene expression in vivo. Although it is unreasonable to expect a permanent transgene expression from an episomal plasmid, long lasting expression even at a low level may offer considerable clinical benefits to gene therapy patients. In addition, viral promoters may not confer tissue-specificity. Since currently the nuclear uptake of delivered DNA is highly inefficient (44) in addition to the low efficiency of liposome-mediated gene expression, no one would worry about the effect of non-specifically expressing a therapeutic gene in vivo. However, when the nuclear uptake and liposome-delivery technology are improved, this has to be seriously considered because there must be an advantage for nature to select genes, such as the cystic fibrosis transmembrane conductance regulatory gene (CFTR), to be epithelium-specific.
If human DNA regulatory elements could direct tissue-specific expression of therapeutic genes at a comparable level to that from strong viral promoters in lung epithelial cells, and sustain gene expression longer than the viral promoters, it would be advantageous to use them for lung gene therapy. At present, there is no suitable expression vector for epithelial tissue gene therapy. There is a need to develop gene therapy cassettes that use human DNA regulatory elements which naturally express genes in epithelial cells and can be used to direct the expression of therapeutic genes. It would be particularly useful if there was an expression cassette that could direct a high level of reporter gene expression in vivo and in vitro. The expression cassette should be safe and confer an appropriate level of tissue-specific expression for a reasonable duration. The expression cassette should be capable of use in epithelial cells, such as submucosal cells.